Creating biomolecules capable of performing any desired function in human cells and animal models represents an interdisciplinary grand challenge critical for rapid progress in biomedical research. The most simple and reliable modern solution remains directed evolution. Unfortunately, the promise of directed evolution for biomedical applications has never been fully realized. The profound challenge is that current directed evolution workflows involve mutagenesis and selection either in vitro, in bacteria, or in yeast; these environments differ greatly from the sophisticated cellular environments of higher eukaryotic disease model systems where the evolved biomolecules must be applied. Consequently, the products of directed evolution often fail to function in biomedically relevant systems. High impact biomedical applications of evolved biomolecules therefore require a re-imagining of current directed evolution platforms. A platform for continuous directed evolution in human cells would have broad impact, allowing the robust evolution of potent monobodies, aptamers, transcription factors, enzymes, and beyond that would be virtually guaranteed to function in biomedically relevant systems. Here, the development and application of a humanized directed evolution platform that permits rapid mutagenesis and efficient selection of active biomolecule variants in human cells is proposed. New methods will be developed to enable the necessary high mutation rates in the context of a human cell, and strategies for both positive and negative selection will be deployed. The use of suspension cells and a bioreactor permit evolutionary workflows on an acceptable laboratory timescale with minimal user intervention. Furthermore, the methods developed will also be readily adaptable for discontinuous directed evolution in human cells. Using this proposed platform, any genetically-encodable biomolecule can be evolved in human cells via a variety of appropriately selected positive and negative selection strategies. The platform will be made broadly available to the research community. Directed evolution experiments during the grant period will focus on high impact functions that cannot be reliably evolved in bacteria or yeast. These include the evolution of functional monobodies, protein-protein interaction inhibitors, RNA aptamers, and novel transcription factors. Additional experiments will explore mutation-driven oncogenesis, cancer cell drug-resistance onset, and the influences of the human proteostasis network on protein evolution during oncogenesis and drug-resistance development. The platform and the research findings are expected to have a broad and deep impact on biomedical research.